A novel approach for chemically deglycosylating O-linked glycoproteins. The deglycosylation of submaxillary and respiratory mucins.

A new approach for removing O-glycosidically linked carbohydrate side chains from glycoproteins is described. Periodate oxidation of the C3 and C4 carbons in peptide-linked N-acetylgalactosamine (GalNAc) residues generates a dialdehyde product which, under mild alkaline conditions, undergoes a beta-elimination which releases carbohydrate and leaves an intact peptide core. The pH and time dependence, and intermediates of the elimination, have been extensively followed by carbon-13 NMR spectroscopy and amino acid analysis using ovine submaxillary mucin (OSM) as the substrate. The deglycosylation of OSM is complete and provides apomucin in high yield with an amino acid composition identical to the starting material. Carboxymethylated OSM when deglycosylated by this method gives an apomucin with an apparent molecular weight of ca. 700 x 10(3). The molecular weight is the same as that calculated for the peptide core of the starting mucin, demonstrating the absence of peptide core cleavage. This contrasts with the use of trifluoromethanesulfonic acid (TFMSA), which generates apomucin products of lower molecular weights. Oligosaccharide side chains substituted at C3 of the peptide-linked GalNAc residue are resistant to the oxidation and elimination. Glycoproteins containing these more complex side chains can be deglycosylated by pretreatment with TFMSA under mild (0 degree C) conditions, which removes peripheral sugars (while leaving the peptide-linked GalNAc residue intact), followed by oxidation and beta-elimination. Studies on the deglycosylation of porcine submaxillary mucin and human tracheobronchial mucin indicate that this approach provides more efficient removal of carbohydrate and less peptide core degradation than a more vigorous (25 degrees C) treatment with TFMSA alone. 13C NMR spectroscopic studies and carbohydrate analysis of the deglycosylation intermediates of the human mucin indicate that certain sialic acid containing and N-acetylglucosamine-containing oligosaccharides have elevated resistance to TFMSA treatment at 0 degrees C. By the use of neuraminidase, repeated mild TFMSA treatments, and multiple oxidations and beta-eliminations, the human mucin can be nearly completely deglycosylated. It is expected that all mucins and most glycoproteins containing O-glycosidic linkages can be readily and nearly completely deglycosylated using this combined approach.     

A chemical method for the deglycosylation of proteins

A simple and rapid chemical method for the deglycosylation of glycoproteins has been developed. The method involves the incubation of protein with trifluoromethanesulfonic acid at 0 degrees C from 0.5 to 2 h followed by the neutralization of the acid with aqueous pyridine at -20 degrees C. The method has been applied effectively to fetuin, ovine submaxillary mucin, ovine lutropin, and human choriogonadotropin. In 1 h almost all of N- and O-linked carbohydrates from ovine lutropin and human choriogonadotropin, with the exception of the linkage N-acetylglucosamine or N-acetylgalactosamine, were removed. Similarly, in 1 h all N-linked carbohydrates, excepting again the linkage sugar, in fetuin were degraded. Longer reaction times up to 2 h completely removed the O-linked carbohydrate chains from fetuin and ovine submaxillary mucin. The deglycosylated hormones thus prepared retained their immunological and biological activities.     

Methylated messenger RNA in mouse kidney.

Polyadenylated messenger RNA from mouse kidney labeled in vivo exhibited a pattern of methylation distinct from that of rRNA and tRNA. After mice were given L-[methyl-3H]methionine, 4% of the polyribosomal RNA label was bound to oligo (dT)-cellulose; 20-24% of orotate- or adenine-labeled polyribosomal RNA eluted in the poly(A)+ RNA fraction under similar conditions. [3H]Methyl radioactivity was not incorporated into low molecular weight (5-5.8 S) rRNA, indicating the extent of nonmethylpurine ring labeling was negligible. [3H]Methyl-labeled poly(A)+ RNA sedimented heterogeneously in sodium dodecyl sulfate containing gradients similarly to poly(A)+ mRNA labeled with [3H]orotic acid. Based on an average molecular length of 2970 nucleotides, renal mRNA was estimated to contain 8.6 methyl moieties per molecule. Analysis of alkaline-hydrolyzed RNA sampled by DEAE-Sephadex-urea chromatography provided estimates of the relative amounts of base and ribose methylation. Although 83% of the [3H]methyl radioactivity in rRNA was in the 2'-0-methylnucleotide fraction, no methylated dinucleotides were found in mRNA. In poly(A)+ mRNA 60% of the [3H]methyl label was in the mononucleotide fraction; the remainder eluted between the trinucleotide and tetranucleotide markers and had a net negative charge between -4 and -5. The larger structure, not yet charcterized, could result from two or three consecutive 2'-0-ribose methylations and is estimated to contain 2.6 methyl residues. Alternatively, the oligonucleotide could be a 5'-terminal methylated nucleotide species containing 5'-phosphate(s) in addition to the 3'-phosphate moiety resulting from alkaline hydrolysis. Either structure could have a role in the processing or translation of mRNA in mammalian cells.     

Translation of partially purified poly(A)+ protamine messenger RNA components in wheat germ and rabbit reticulocyte cell-free systems. Evidence for translational control mechanisms.

The coding properties of individual poly(A)+ protamine mRNA subcomponents have been explored by analysis of their translation products in two different cell-free protein synthesis systems, the rabbit reticulocyte lysate and the wheat germ S-30, both of which can translate total protamine mRNA. The products synthesized in the reticulocyte lysate in the presence of total poly(A)+ PmRNA consisted mainly of protamine components CII and CIII with component CI only a minor product. However, in the wheat germ S-30, the same mRNA preparation supported the synthesis of all three protamine components, in approximately equal amounts. In addition a new polypeptide, a putative fourth protamine component, labelled CO, was also synthesized. The translation products of subcomponents of poly(A)+ PmRNA separated as individual bands on polyacrylamide gels were similarly analyzed and it was shown that each of the isolated poly(A)+ PmRNA species could stimulate the incorporation of [3H]arginine into protamines in both translational systems. Although each mRNA band stimulated the synthesis of one particular protamine polypeptide predominantly in a given cell-free system, the same RNA preparation was found to direct preferentially the synthesis of a different protamine component in the second cell-free system. The products synthesized in the rabbit reticulocyte lysate in the presence of the individual mRNA species still showed component CI present as a minor product.     

Characterization of mucin glycoprotein-specific translation products from swine and human trachea,pancreas and colon

Summary RNA was isolated from cultured swine trachea epithelial cells and mucus-secreting tumor cell lines from human pancreas, lung and colon by extraction with guanidine isothiocyanate. Poly(A)⁺mRNA rich fractions were purified by repeated chromatography on oligo (dT)-cellulose columns and they were translated in a cell-free rabbit reticulocyte system. Translation products labelled with ³⁵S-methionine were isolated by immunoprecipitation with specific antibodies to the polypeptide chains of mucin glycoproteins and they were analyzed by SDS-PAGE and fluorography. A single principal polypeptide band of 67 kDa was found in all cases when the immunoprecipitates were washed with buffer containing bovine serum albumin and unlabeled deglycosylated mucin glycoprotein. The intensity of the 67 kDa band decreased when unlabeled deglycosylated mucin glycoprotein was added to the translation mixture before immunoprecipitation. Affinity purified monospecific antibodies elicited against chemically deglycosylated polypeptide chains of purified mucin glycoproteins from human and swine trachea and Cowper's gland were all equally effective in immunoprecipitating the 67 kDa translation product. Monospecific antibodies directed against the glycosylated and unglycosylated regions of the polypeptide chain yielded single bands with a molecular size of 67 kDa in each case. Peptide profiles obtained by digestion of the 67 kDa translation product with S. aureus V-8 protease were identical to those obtained with deglycosylated human and swine trachea mucin glycoproteins.These stydies clearly demonstrate that the translation product of swine trachea and human lung, colon and pancreatic mucin glycoprotein gene is a single polypeptide chain of 67 kDa. The relative size and properties of the translation products synthesized with poly (A)⁺RNA isolated from mucus-secreting cells derived from three different tissues are similar to those of mucin glycoproteins purified directly from mucus secretions of human and swine trachea epithelium.Abbreviations TFMS Trifluoromethanesulfonic acid - SDS Sodium Dodecyl Sulfate - PAGE Polyacrylamide Gel Electrophoresis - GalNAc N-Acetylgalactosamine - HTMG Human Trachea Mucin Glycoprotein - deHTMG deglycosylated Human Trachea Mucin Glycoprotein - STMG Swine Trachea Mucin Glycoprotein - deSTMG deglycosylated Swine Trachea Mucin Glycoprotein - CCMG Cowper's Gland Mucin Glycoprotein - deCGMG deglycosylated Cowper's Gland Mucin Glycoprotein - HPMG Pancreatic Mucin Glycoprotein from BxPC-3 cells - HCMG Colon Mucin Glycoprotein from SW 403 cells - HLMG Human Lung Mucin Glycoprotein from A-549 cells - STMG⁺deSTMG^– antibodies which bind to immobilized STMG but do not bind to immobilized deSTMG - deSTMG⁺STMG^– antibodies which bind to immobilized deSTMG but do not bind to immobilized STMG - STMG⁺deSTMG⁺ antibodies which bind to both STMG and deSTMG - HTMG⁺deHTMG^– antibodies which bind to immobilized HTMG but do not bind to immobilized deHTMG - deHTMG⁺HTMG^– antibodies which bind to immobilized deHTMG but do not bind to immobilized HTMG - HTMG⁺deHTMG⁺ Antibodies which bind to both HTMG and deHTMG     

Molecular cloning of human intestinal mucin cDNAs. Sequence analysis and evidence for genetic polymorphism

A human small intestine lambda gt11 cDNA library was screened using antisera prepared against the deglycosylated protein backbone of human colon cancer xenograft mucin. Three cDNAs were isolated from this screening, designated SMUC 40-42. These cDNAs were all found to contain tandem repeats of 69 nucleotides which encoded a threonine- and proline-rich protein consensus sequence of PTTTPITTTTTVTPTPTPTGTQT. RNA blots probed with one of these cDNAs, SMUC 41, exhibited large, polydisperse hybridization bands at approximately 7,600 bases. Band intensities were strongest when human small intestine, colon, and colon cancer poly(A)+ RNA was used. In vitro translation of poly(A)+ RNA from human small intestine, colon, and colon cancer cells produced a 162,000-dalton peptide that was immunoprecipitated with antibodies to deglycosylated mucin. SMUC 41 was also used to probe DNA blots, which indicated the presence of restriction fragment length polymorphisms in the intestinal mucin gene. These findings may be important in assessing the abnormal mucins found associated with several human diseases.     

Gene expression and cDNA cloning identified a major basic protein constituent of bovine seminal plasma as bovine monocyte-chemoattractant protein-1 (MCP-1).

P6 is one of the major basic proteins of bovine seminal plasma. Using cell-free translation of poly(A)+RNA from bovine seminal vesicle tissue and monospecific anti-P6-IgGs, we show that P6 is a secretory product of the seminal vesicles. Immunohistochemical experiments supported this finding. Immunoscreening of a lambda gt11 cDNA library derived from seminal vesicle poly(A)+RNA furnished a number of positive cDNA clones, from which clone pH42 was characterized by sequencing. The partial amino acid sequence of a CNBr-fragment of P6 permitted identification of the reading frame of clone pH42 encoding the precursor protein of P6. The P6 precursor contains a signal peptide of 23 amino acids followed by the mature P6 sequence of 76 amino acid residues. The cDNA sequence of pH42 was 80% homologous with that of the human monocyte-chemoattractant protein-1 (hMCP-1). The respective amino acid sequences for the precursor molecules are 72% identical. Northern analysis of seminal vesicle poly(A)+RNA using pH42 as probe probe identified a 0.9-kb P6 mRNA. Stimulation of P6 mRNA expression by phytohemagglutinin in bovine peripheral mononuclear leukocytes suggests that P6 is identical to bovine MCP-1.     

Isolation and characterization of Dorin M, a lectin from plasma of the soft tick Ornithodoros moubata

A lectin with high hemagglutinating activity, which we have named Dorin M, was identified in the plasma of the soft tick Ornithodoros moubata. The activity of the plasma lectin could be efficiently inhibited by sialic acid, N-acetyl-D-hexosamines and sialoglycoproteins. Dorin M was purified to homogeneity using two different isolation systems: affinity chromatography on a column of bovine submaxillary mucin conjugated to Sepharose 4B with specific elution by N-acetyl-D-glucosamine and chromatography on Blue-Sepharose followed by anion exchange FPLC on a MonoQ column. The purified lectin is a glycoprotein which, in the native state, forms aggregates with molecular mass of about 640 kDa. Non-reducing SDS PAGE revealed that the lectin consists of two noncovalently bound subunits migrating closely around 37 kDa. Dorin M is a glycoprotein, probably modified by N-type glycosylation. After chemical deglycosylation, only one band of about 32 kDa was detected. Dorin M is the first lectin purified from ticks.     

Mucin biosynthesis. Characterization of UDP-galactose: alpha-N-acetylgalactosaminide beta 3 galactosyltransferase from human tracheal epithelium

We have characterized the UDP-galactose: alpha-N-acetylgalactosaminide beta 3 galactosyltransferase in human tracheal epithelium using asialo ovine submaxillary mucin as the acceptor. Maximal enzyme activity was obtained at pH 6.0-7.5 and at 20-25 mM MnCl2 and at 2% Triton X-100. Cd2+ could substitute for Mn2+ as the divalent ion cofactor. Spermine, spermidine, putrecine, cadaverine, and poly-L-lysine stimulated the enzyme activity at low (2.5 mM) MnCl2 concentration. The apparent Michaelis constants for N-acetylgalactosamine, asialo ovine submaxillary mucin, and UDP-galactose were 15.5, 1.14, and 1.36 mM, respectively. The enzyme activity was not affected by alpha-lactalbumin. The alpha-N-acetygalactosaminide beta 3 galactosyltransferase was shown to be different from the N-acetylglucosamine galactosyltransferase by acceptor competition studies. The product of galactosyltransferase was identified as Gal beta 1 leads to 3GalNAc alpha Ser (Thr) by (a) isolation of [14C]Gal-GalNAc-H2 after alkaline borohydride treatment of the 14C-labeled product, (b) establishment of the beta-configuration of the newly synthesized glycosidic bond by its complete cleavage by bovine testicular beta-galactosidase, and (c) assignment of the 1 leads to 3 linkage by identification of threosaminitol obtained from the oxidation of the disaccharide with periodic acid followed by reduction with sodium borohydride, hydrolysis in 4 N HCl, and analysis on an amino acid analyzer. The 1 leads to 3 linkage was confirmed by its resistance to jack bean beta-galactosidase and by the presence of a m/e 307 ion fragment and the absence of a m/e 276 ion by gas-liquid chromatography-mass spectrometry analysis. When acid and beta-galactosidase-treated human tracheobronchial mucin was used as the acceptor, 3.3% of the product was found as [14C]Gal-GalNAc-H2. The remainder of the [14C]Gal was found in longer oligosaccharides formed by a different beta-galactosyltransferase. This galactosyltransferase is slightly inhibited by alpha-lactalbumin and stimulated by spermine.     

Streptococcal Sialidase I. Isolation and Properties of Sialidase Produced by Group K Streptococcus

A survey has been made of the activity of a wide variety of standard strains of streptococci against bovine submaxillary mucin. Strain 6646 (group K) and strain D 168A "X" (group M) completely broke down and strain H 60R (group F) incompletely broke down bound sialic acid of bovine submaxillary mucin added to the growth medium. Among these strains, strain 6646 (group K) produced sialidase in the cell and in the culture fluid. An appropriate amount of glucose in the culture medium stimulated growth and the production of enzyme, but an excess of glucose in the culture medium caused abundant growth without production of the enzyme. The streptococcal sialidase was precipitated from the culture fluid by ammonium sulfate at 50% saturation, and further purification was achieved by diethylaminoethyl cellulose chromatography. Ca(++) and Co(++) stimulated the sialidase activity, and Mn(++), Zn(++), and ethylenediaminetetraacetate inhibited it. With acetate buffer, the optimal pH lay between 5 and 6. Sialic acid was detected in the reaction product of the streptococcal sialidase and bovine submaxillary mucin.     

Molecular cloning of the carboxy terminus of a canine tracheobronchial mucin.

A cDNA library constructed from canine tracheal mRNA was screened with polyclonal antiserum specific to canine tracheal apomucin (CTM-A). Eight antibody reactive clones were isolated and purified to clonality. One of the clones, designated pCTM-A, had a 1.7 kb insert and included a single open reading frame with a poly (A)+ tail. The amino acid composition of the encoded protein was consistent with that expected for CTM-A. The fusion protein produced by cloning the 1.7 kb insert in the pMALc expression vector reacted with the purified anti-apomucin CTM-A antibody. Also, polyclonal antibodies raised to the purified protein product encoded by pCTM-A reacted with deglycosylated CTM-A confirming that this clone does indeed code for apomucin CTM-A. This is the first report of a cDNA encoding the C-terminus of a canine tracheal mucin.     

Analysis of mucin-derived oligosaccharides by medium-pressure gel-permeation and amino-plate thin-layer chromatography conducted in conjunction

Oligosaccharides present in mucin were labeled by reduction with NaB3H4 and separated by gel-permeation chromatography with a Toyopearl HW-40S column using 0.1 M pyridine acetate, pH 5.0, as the solvent. Each fraction was further analyzed by thin-layer chromatography (TLC) on a Funagel AMP plate, a glass plate precoated with 3-aminopropyl-bonded silica. Acetonitrile/10 mM triethylamine acetate (3/2, by volume) served as the solvent. The sites of oligosaccharides on the TLC plate could be determined according to size, anionic charge, and sugar composition. They could thus be "mapped" on the plate. In this manner, the distribution of oligosaccharides on bovine submaxillary mucin and rat gastric mucin was determined. Each radiolabeled oligosaccharide in newly synthesized rat gastric mucin, metabolically labeled with [14C]glucosamine or [35S]sulfate, was also identified by this method.     

Thyroxine increases neonatal mouse submandibular gland mRNA-directed synthesis of epidermal growth factor

Thyroid hormones are known to modulate the concentrations of epidermal growth factor (EGF) in the mouse submandibular gland (SMG); this action is presumably mediated by the nuclear triiodothyronine receptor. To test the hypothesis that thyroid hormones act to increase SMG EGF concentrations by increasing the number of poly(A)+ -specific mRNA, poly(A)+ RNA was isolated from SMGs of neonatal mice which had been treated daily from birth through to 21 days of age with thyroxine (T4,0.4 microgram/g body weight). Poly(A)+ RNA also was extracted from SMGs of intact 21-day-old mice which had received vehicle alone. No significant differences in total nucleic acid, total RNA, or poly(A)+ RNA yields were noted between the two groups of animals. The isolated poly(A)+ RNAs from T4-treated and control mice were translated in an in vitro wheat germ system. Although no significant differences in efficiency of [35S]cysteine incorporation into trichloracetic acid precipitable material were noted between the two poly(A)+ RNA preparations, a significantly greater proportion of radioactivity was immunoprecipitable by anti-EGF antiserum in the translation medium derived from T4-treated mice (17.2 +/- 0.9%, mean +/- SEM) than in that of control mice (7.3 +/- 0.5%, P less than 0.001). Polyacrylamide gel electrophoresis of the immunoprecipitates (IMMP) revealed the presence of three radioactive bands with apparent relative masses (MrS) of 12,000, 9000, and 6000. The latter species comigrated with purified EGF, [125I]EGF, and an IMMP of a SMG extract. The translation product IMMPs following polyacrylamide gel electrophoresis were iodinated and digested with alpha-chymotrypsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of the carbohydrate moiety in thyrotropin action

The relative binding affinity of deglycosylated human TSH was 6-fold higher than that of native TSH. Although deglycosylated human TSH significantly stimulated adenylate cyclase, it was less effective than the native hormone. When deglycosylated human TSH was added with bovine TSH, however, a dose-dependent antagonism was observed. In particular, submaximal and maximal concentrations of bovine TSH and deglycosylated human TSH resulted in cAMP values much lower than the sum of activities of the individual hormones. The data suggest that although the effects of TSH deglycosylation are not as dramatic as with the gonadotropins, the carbohydrates of TSH appear to be required for maximal activation of adenylate cyclase by the hormone.     

Use of active site-directed inhibitors to study in situ degradation of glycoproteins by the perfused rat liver

Use was made of the asialoglycoprotein receptor system in a perfused rat liver in order to study lysosomal degradation and subsequent metabolism of radioactive derivatives of asialo-ovine submaxillary mucin and asialo-alpha 1-acid glycoprotein. A trace of N-acetyl-D-[6-3H]galactosamine-labeled asialo-ovine submaxillary (4 micrograms) was completely taken up by the tissue in less than 20 min. After 3 h 24% of the radioactivity from the mucin reappeared on newly synthesized serum glycoproteins that were secreted into the perfusate. [6-3H] Galactose asialo-alpha 1-acid glycoprotein was also rapidly cleared by the liver; however, after 3 h greater than 60% of the radioactivity derived from this sugar labeled glycoprotein was secreted back into the perfusate as [3H]glucose. Rat livers perfused with 0.15 mM beta-D-galactopyranosylmethyl-p-nitrophenyltriazene lost 90% of their beta-D-galactosidase activity within 1 h while other representative glycosidases showed no change as followed by hydrolysis of p-nitrophenylglycosides. Livers pretreated with this triazene compound metabolized [3H]GalNAc asialo-ovine submaxillary mucin normally but were unable to process [3H]Gal asialo-alpha 1-acid glycoprotein as evidenced by a complete inhibition of [3H]glucose release following addition of the latter substrate. Metabolism of N-acetyl[14C]glucosamine asialo-alpha 1-acid glycoprotein was similarly inhibited by 70%. 125I-labeled asialo-alpha 1-acid glycoprotein catabolism was not affected by the chemically induced beta-D-galactosidase deficiency. Subcellular fractionation of inhibitor-treated livers accumulating radioactive carbohydrate showed the majority of the label was associated with a fraction enriched in lysosomes. Analysis of the trapped radioactivity by high resolution Bio-Gel P-4 chromatography revealed nearly intact oligosaccharides minus only the reducing N-acetylglucosamine of the chitobiose core. Direct comparison of these sugar chains with those isolated from human and canine GM1 gangliosidosis liver by silicic acid thin layer chromatography showed those isolated from rat liver to be identical to the major subset of oligosaccharides found in the human disease. In similar experiments in which the galactosyl triazene was replaced by swainsonine, an alpha-D-mannosidase inhibitor, catabolism of [14C]GlcNAc asialo-alpha 1-acid glycoprotein resulted in the accumulation of a single oligosaccharide of the structure. Man3[14C]GlcNAc1. These results demonstrate an endo-N-acetyl-beta-D-glucosaminidase is active in rat liver lysosomes.     

Purification and characterization of a sialic acid specific lectin from the hemolymph of the freshwater crab Paratelphusa jacquemontii.

A naturally occurring hemagglutinin was detected in the serum of the freshwater crab, Paratelphusa jacquemontii (Rathbun). Hemagglutination activity with different mammalian erythrocytes suggested a strong affinity of the serum agglutinin for horse and rabbit erythrocytes. The most potent inhibitor of hemagglutination proved to be bovine submaxillary mucin. The lectin was purified by affinity chromatography using bovine submaxillary mucin-coupled agarose. The molecular mass of the purified lectin was 34 kDa as determined by SDS/PAGE. The hemagglutination of purified lectin was inhibited by N-acetylneuraminic acid but not by N-glycolylneuraminic acid, even at a concentration of 100 mm. Bovine submaxillary mucin, which contains mainly 9-O-acetyl- and 8,9 di-O-acety-N-acetyl neuraminic acid was the most potent inhibitor of the lectin. Sialidase treatment and de-O-acetylation of bovine submaxillary mucin abolished its inhibitory capacity completely. Also, asialo-rabbit erythrocytes lost there binding specificity towards the lectin. The findings indicated an O-acetyl neuraminic acid specificity of the lectin.     

The effect of age on the properties of poly(A)-containing messenger RNA in Physarum polycephalum

The effect of ageing on the properties of polysomal poly(A)-containing messenger RNA [poly(A)+ mRNA] of Physarum polycephalum has been investigated. Using poly(U)--Sepharose affinity chromatography it was shown that shortening of the poly(A) tract occurred as the age of the mRNA population increased. Analysis of the poly(A) segments by use of polyacrylamide gel electrophoresis, after digestion of polysomal poly(A)+ mRNA molecules with RNAase A and RNAase T1, revealed that their lengths ranged from 140 to 220 nucleotide residues. A reduction in the efficiency of utilization of mRNA for translation as the age of the mRNA population increased was demonstrated by measuring the proportion of poly(A)+ mRNA present in the polysomal fraction as compared with post-polysomal material.